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PubMed1975

Chromosomes of a cell line of Dipodomys panamintinus (kangaroo rat). A banding and autoradiographic study.

The chromosomes of an established cell line of Dipodomys panamintinus have been characterised in terms of their C, G and Q banding patterns, and the distributions of silver grains in autoradiographs of chromosomes labelled in early or late S phase. No relationship could be established between C, G or Q banding regions of chromosomes and a particular S phase time of replication of the DNA in these banded regions. The implication of this result to the concept of heterochromatin is discussed.

Animals↗
PubMed1994

Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos.

A novel approach to study the higher level packaging of specific DNA sequences has been developed by coupling high-resolution fluorescence hybridization with biochemical fractionation to remove histones and distend DNA loops to form morphologically reproducible nuclear "halos." Results demonstrate consistent differences in the organization of specific sequences, and further suggest a relationship to functional activity. Pulse-incorporated bromodeoxyuridine representing nascent replicating DNA localized with the base of the chromatin loops in discrete clustered patterns characteristic of intact cells, whereas at increasing chase times, the replicated DNA was consistently found further out on the extended region of the halo. Fluorescence hybridization to unique loci for four transcriptionally inactive sequences produced long strings of signal extending out onto the DNA halo or "loop," whereas four transcriptionally active sequences remained tightly condensed as single spots within the residual nucleus. In contrast, in non-extracted cells, all sequences studied typically remained condensed as single spots of fluorescence signal. Interestingly, two transcriptionally active, tandemly repeated gene clusters exhibited strikingly different packaging by this assay. Analysis of specific genes in single cells during the cell cycle revealed changes in packaging between S-phase and non S-phase cells, and further suggested a dramatic difference in the structural associations in mitotic and interphase chromatin. These results are consistent with and suggestive of a loop domain organization of chromatin packaging involving both stable and transient structural associations, and provide precedent for an approach whereby different biochemical fractionation methods may be used to unravel various aspects of the complex higher-level organization of the genome.

Cell Cycle↗
PubMed2006

Indirect measurement of the lag time distribution of single cells of Listeria innocua in food.

The distribution of log counts at a given time during the exponential growth phase of Listeria innocua measured in food samples inoculated with one cell each was applied to estimate the distribution of the single-cell lag times. Three replicate experiments in broth showed that the distribution of the log counts is a linear mapping of the distribution of the detection times measured by optical density. The detection time distribution reflects the lag time distribution but is shifted in time. The log count distribution was applied to estimate the distributions of the lag times in a liquid dairy product and in liver paté after different heat treatments. Two batches of ca. 100 samples of the dairy product were inoculated and heated at 55 degrees C for 45 min or at 62 degrees C for 2 min, and an unheated batch was incubated at 4 degrees C. The final concentration of surviving bacteria was ca. 1 cell per sample. The unheated cells showed the shortest lag times with the smallest variance. The mean and the variance of the lag times of the surviving cells at 62 degrees C were greater than those of the cells treated at 55 degrees C. Three batches of paté samples were heated at 55 degrees C for 25 min, 62 degrees C for 81 s, or 65 degrees C for 20 s. A control batch was inoculated but not heated. All paté samples were incubated at 15 degrees C. The distribution of the lag times of the cells heated at 55 degrees C was not significantly different from that of the unheated cells. However, at the higher temperatures, 62 degrees C and 65 degrees C, the lag duration was longer and its variance greater.

Animals↗
PubMed2000

Molecular strategies for interrupting arthropod-borne virus transmission by mosquitoes.

Arthropod-borne virus (arbovirus) infections cause a number of emerging and resurgent human and veterinary infectious diseases. Traditional means of controlling arbovirus diseases include vaccination of susceptible vertebrates and mosquito control, but in many cases these have been unavailable or ineffective, and so novel strategies for disease control are needed. One possibility is genetic manipulation of mosquito vectors to render them unable to transmit arboviruses. This review describes recent work to test the concept of pathogen-derived resistance in arthropods by expression of viral genes in mosquito cell cultures and mosquitoes. Sense and antisense genome sequences from La Crosse virus (LAC) (a member of the Bunyaviridae) and dengue viruses serotypes 1 to 4 (DEN-1 to DEN-4) (members of the Flaviviridae) were expressed in mosquito cells from double-subgenomic and replicon vectors based on Sindbis virus (a member of the Togaviridae). The cells were then challenged with homologous or related viruses. For LAC, expression of antisense sequences from the small (S) genome segment, particularly full-length antisense S RNA, effectively interfered with replication of challenge virus, whereas expression of either antisense or sense RNA from the medium (M) segment was completely ineffective in LAC inhibition. Expression of sense and antisense RNA derived from certain regions of the DEN genome also blocked homologous virus replication more effectively than did RNA from other regions. Other parameters of RNA-mediated interference have been defined, such as the time when replication is blocked and the minimum size of effector RNA. The mechanism of RNA inhibition has not been determined, although it resembles double-stranded RNA interference in other nonvertebrate systems. Prospects for application of molecular strategies to control arbovirus diseases are briefly reviewed.

Animals↗
PubMed2006

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus.

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.

Caulobacter crescentus↗
PubMed1987

Changing spatial patterns of DNA replication in the developing wing of Drosophila.

Using an antibody against bromodeoxyuridine we have analyzed the distribution of S-phase nuclei in the wing disc of Drosophila as the larval disc transforms into the adult wing during metamorphosis. On the basis of the timing of replication three cell populations can be distinguished: the cells of the presumptive wing margin, the precursor cells of the longitudinal veins, and those of the intervein regions. In each of these populations the cell cycle is first arrested and later resumes at a specific time, so that at each developmental time point a characteristic spatial pattern of S-phase nuclei is seen. An interpretation of these changing patterns in terms of vein formation, compartments, and neural development is offered.

Animals↗
PubMed2000

Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells.

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.

Animals↗
PubMed1988

Timing of proto-oncogene replication: a possible determinant of early S phase sensitivity of C3H 10T1/2 cells to transformation by chemical carcinogens.

The temporal order of replication of several genes was studied in 10T1/2 cells synchronized by release from confluence-induced arrest of proliferation followed by treatment with 2 micrograms/mL aphidicolin for 24 h. DNA subjected to bromodeoxyuridine substitution for 1- or 2-h intervals spanning the S phase was separated from the remaining DNA in cesium chloride gradients, filtered onto nitrocellulose in a slot-blot apparatus, and hybridized with various 32P-labeled probes. Ha-ras was among the first genes replicated at the onset of the S phase. The myc proto-oncogene replicated later but within the first hour of the S phase. The replication of Ki-ras, raf, and mos was detected between hour 1 and 2 of the S phase. The dihydrofolate reductase gene replicated early (0-2 h) and the myb proto-oncogene replicated in mid-S phase (2-4 h). An immunoglobulin VH sequence and the beta-globin gene replicated late in 10T1/2 cells, 4-6 h after removal of aphidicolin. Replicating DNA is preferentially adducted by chemical carcinogens, and replication of damaged proto-oncogenes before they are repaired may activate their transforming potential. Therefore, the observed replication of proto-oncogenes during the early S phase may underlie the enhanced sensitivity of 10T1/2 cells to chemically induced transformation at this point in the cell cycle.

Animals↗
PubMed1993

The role of lymphoid organs in the pathogenesis of HIV infection.

Following primary human immunodeficiency virus (HIV) infection, HIV disease is characterized by a prolonged period, usually lasting several years, of clinical latency. During this period viremia is generally very low or undetectable, the number of infected cells (i.e. viral burden) in the blood are very low, and the levels of viral replication in these cells are barely detectable. These findings have been interpreted as a reflection of a phase of inactive HIV disease during which time HIV replicates very slowly or its replicating ability is kept under control by effective HIV specific immune responses. However, during this period a general deterioration of immune function and progressive depletion of CD4+ T cells occur; the inevitable outcome is clinically apparent disease. In the present article, we describe a model of disease development in which HIV infection is both active and progressive in the lymphoid organs during the clinically latent period of HIV infection when there are few, if any, signs of disease activity in peripheral blood.

CD4-Positive T-Lymphocytes↗
PubMed2005

Chromosome and replisome dynamics in E. coli: loss of sister cohesion triggers global chromosome movement and mediates chromosome segregation.

Chromosome and replisome dynamics were examined in synchronized E. coli cells undergoing a eukaryotic-like cell cycle. Sister chromosomes remain tightly colocalized for much of S phase and then separate, in a single coordinate transition. Origin and terminus regions behave differently, as functionally independent domains. During separation, sister loci move far apart and the nucleoid becomes bilobed. Origins and terminus regions also move. We infer that sisters are initially linked and that loss of cohesion triggers global chromosome reorganization. This reorganization creates the 2-fold symmetric, ter-in/ori-out conformation which, for E. coli, comprises sister segregation. Analogies with eukaryotic prometaphase suggest that this could be a primordial segregation mechanism to which microtubule-based processes were later added. We see no long-lived replication "factory"; replication initiation timing does not covary with cell mass, and we identify changes in nucleoid position and state that are tightly linked to cell division. We propose that cell division licenses the next round of replication initiation via these changes.

Chromosome Mapping↗
PubMed1986

Timing of initiation of chromosome replication in individual Escherichia coli cells.

The synchrony of initiation of chromosome replication at multiple origins within individual Escherichia coli cells was studied by a novel method. Initiation of replication was inhibited with rifampicin or chloramphenicol and after completion of ongoing rounds of replication the numbers of fully replicated chromosomes in individual cells were measured by flow cytometry. In rapidly growing cultures, with parallel replication of several chromosomes, cells will end up with 2n (n = 1, 2, 3) chromosomes if initiation occurs simultaneously at all origins. A culture with asynchronous initiation may in addition contain cells with irregular numbers (not equal to 2n) of chromosomes. The frequency of cells with irregular numbers of chromosomes is a measure of the degree of asynchrony of initiation. After inhibition of initiation and run-out of replication in rapidly growing B/r A and K-12 cultures, a small fraction of the cells (2-7%) contained 3, 5, 6 or 7 chromosomes. From these measurements it was calculated that initiation at four origins in a single cell occurred within a small fraction, 0.1, of the doubling time (tau). A dnaA(Ts) mutant strain grown at permissive temperature exhibited a very large fraction of cells with irregular numbers of chromosomes after drug treatment demonstrating virtually random timing of initiation. A similar pattern of chromosome number per cell was found after treatment of a recA strain.

Chloramphenicol↗
PubMed1994

Enzyme-dependent pausing during in vitro replication of O4-methylthymine in a defined oligonucleotide sequence.

We had previously reported that an oligonucleotide containing a site-specifically incorporated O4-methylthymine (m4T) was replicated under kinetic conditions by the Klenow fragment of E. coli DNA polymerase I (Kf) (Dosanjh et al., 1993). Using other polymerases for complete replication, but with limiting enzyme, a pause site before the m4T was observed. In order to investigate whether such a pause could be due to enzyme dissociation or stalling, trapping experiments were designed to aid in differentiating the two mechanisms. Rather than the generally used heparin or sheared DNA trap, these experiments utilized as the acceptor the same oligonucleotide containing unmodified thymine. It was observed that, under enzyme-limiting conditions, the nature of the enzyme played a major role in replication of m4T. With a running start, Kf and calf-thymus polymerase alpha-primase allowed replication beyond the m4T, while Sequenase and T7 showed a strong pause site at the base before m4T. When the oligonucleotide trap was added after different times of replication, it was found that Sequenase remained bound to the template-primer, regardless of whether T or m4T was present. In contrast, Kf dissociated and re-associated rapidly. Thus, m4T appears to be a strong replication block when using limiting amounts of a highly processive enzyme such as Sequenase or T7. This may imply that such enzymes discriminate against forming a poor basepair but remain bound to the primer-template or become inactivated.

Base Sequence↗
PubMed1991

Replication of ocular isolates of human adenovirus is serotype-dependent in rabbit corneal organ culture.

The goal of the present in vitro study was to determine the ability of unadapted human adenoviral ocular isolates to replicate in the rabbit cornea. Rabbit corneas grown in organ culture (24 well plate) were inoculated topically with 50 microliters (5 x 10(5) pfu) of different ocular adenoviral serotypes (ATCC and clinical isolates). Control wells (no cornea present) were inoculated in a similar fashion. Viral replication was determined by serial aliquots titrated on A549 cells. We demonstrated sustained viral replication over time of all isolates (100%) of Ad1, 2, 5, 6, 8, 9, 11 and 37 tested. No isolates (0%) of Ad3, 7A, 19, and 4 demonstrated replication in our model. Peak titers varied among successful serotypes from 10(2) pfu/ml (Ad11) to 10(5) PFU/ml (Ad5), and among different isolates of a given serotype. We conclude that the ability of unadapted human Ad serotypes to replicate in rabbit corneas was serotype-dependent, and that subgroup C (Ad1, 2, 5, and 6) appeared to be the most successful subgroup.

Adenoviruses, Human↗
PubMed1997

Replicative senescence and cell immortality: the role of telomeres and telomerase.

Telomere shortening is correlated with cell senescence in vitro and cell aging in vivo. The telomere hypothesis suggests that telomere length serves as a mitotic clock for timing cellular replicative life span. Expression of telomerase stabilizes telomere length and allows for continual replication, or cell immortality. This article reviews recent evidences for the role of telomere length and telomerase in the regulation of cellular replicative life span. The therapeutic potential of manipulating telomerase expression and telomere length is also discussed.

Animals↗
PubMed1983

Sequential mutagenesis of drug resistance in Streptococcus mutans during synchronous replication.

The frequency of nitrosoguanidine-induced mutation for streptomycin-, bacitracin- and rifampicin-resistance was measured in Streptococcus mutans during synchronous replication after release from chloramphenicol inhibition. A clear peak of mutagenesis for each marker was observed at certain times during synchronous replication. These times were different for individual markers; the times of peaks for streptomycin-, bacitracin- and rifampicin-resistance were 13, 22 and 12 min, respectively. At a definite time after the first peak, there was a second one. The distances between the first and second peaks during the synchronous replication were identical for all markers and approximately 50 min which represents the doubling time of the organisms. These results indicate that nitrosoguanidine causes sequential mutagenesis for these three markers in Streptococcus mutans when the growth is resumed after chromosome alignment, so that the methods may be useful in determining the sequence of gene replication for various markers in Streptococcus mutans.

Anti-Bacterial Agents↗